B cells are an important pathogenic component of autoimmunity. However, our current knowledge of the phenotype of pathogenic B cells in these diseases is still limited and more studies are needed to further characterize these cells and uncover their unique characteristics and mode of action. B cells are known to express only one type of antibody and be specific for one antigen. However, rare B cells co-expressing two different antibodies (i.e., dual-reactive B cells) exist in mice and are present in humans. Our goal is to understand whether dual-reactive B cells represent a relevant B cell subset in autoimmunity. Our novel findings demonstrate that autoimmune mice generate dual-reactive immature and mature B cells more frequently than nonautoimmune mice. The majority of these cells react with self-antigens indicating that they evade mechanisms of negative selection. Dual-reactive B cells generate autoantibodies more frequently than single-reactive B cells and are highly enriched in the antigen-activated B cell subsets of autoimmune mice. Finally, we have identified a molecular pathway for the positive selection of nonautoreactive immature B cells that we propose is used for the generation of dual-autoreactive B cells as well. In the previous grant cycle, we have established the bases for the proposed research by creating the mutant mice and methodologies with which to follow development and selection of dual-reactive B cells in autoimmune and nonautoimmune mice. Therefore, we are uniquely posed to carry out the proposed studies. Our innovative hypothesis is that dual-reactive B cells can evade mechanisms of B cell tolerance to become an important component of autoimmune diseases. The goal of the proposed research is to deepen our understanding of the relationship between dual-reactive B cells and autoimmunity in mice. We will investigate the mechanisms that cause increased generation, selection and enrichment of dual-reactive B cells in autoimmune-prone mice and whether these B cells contribute to the development of autoimmunity. We will also translate our mouse studies by examining the prevalence of dual-reactive B cells in individuals with autoimmunity. To achieve our goals we will develop the following specific aims: 1) To determine whether tonic B cell receptor signaling inhibits receptor editing in dual-autoreactive immature B cells and promotes their differentiation via the Ras-Erk pathway; 2) To establish some of the mechanisms of antigen-mediated selection and activation of dual-reactive B cells in autoimmune mice; 3) To determine whether dual-reactive B cells contribute to autoimmunity and are enriched in autoimmune patients. The studies proposed here will establish whether dual-reactive B cells are a diagnostic and/or pathogenic B cell subset in autoimmunity and will be of value for the development of methods to identify and target these B cells. Overall, these studies are important for understanding B cell-mediated mechanisms of autoimmune development and to uncover novel targets for autoimmune intervention.